The goal of this research project is to structurally characterize the origins of replication of the eucaryotic slime mold Physarum polycephalum, the bacteriophage T4, and the bacterium Escherichia coli: and to study the mechanisms by which replication is initiated at these origins. Several DNA fragments which are replicated at the start of S phase in Physarum will be selected from a library of the entire Physarum genome cloned in the phage lambda derivative Charon 4A. Segments of 100 to 200 kilobase pairs which surround these early replicating fragments will be physically mapped and then the temporal pattern of replication within these segments will be followed by hybridization to radioactive DNA isolated from macroplasmodia of Physarum pulse-labeled at intervals during the S phase. This should allow us to more clearly define the manner in which replication is controlled throughout the S period in lower eucaryotes. In the case of T4, restriction endonucleases will be used to dissect partially replicated chromosomes and provide defined ends from which to pinpoint by electron microscopy the location of the T4 origin(s) of replication. An attempt will be made to clone the E. coli origin of replication, oriC, on a plasmid which is temperature sensitive for initiation of this replication. This should produce a hybrid plasmid capable of replication either from the E. coli origin or the endogenous origin of the plasmid. With this recombinant plasmid, it should then be possible to select and maintain E. coli oriC mutants for structural characterization.